Alcohol dependence and HCV: mechanisms of combined CNS injury

描述(由申请人提供)： 项目摘要长期饮酒在大脑中的主要后果之一是炎症增加，导致神经退化，并伴随着相关的认知和精神障碍。药物滥用后的患者持续存在神经精神障碍，并与较差的治疗结果有关。丙型肝炎病毒还与多种肝外综合征有关，包括中枢神经系统(CNS)损害和神经精神障碍。然而，目前尚不清楚这种认知和情绪障碍是系统性疾病、肝功能受损还是中枢神经系统病毒感染所致。需要动物模型来提供新的见解，以了解酒精依赖和慢性病毒感染共病影响的分子机制和途径，以及那些在戒酒和病毒清除后持续存在的神经精神损害的原因。我们的总体假设是，合并AUDS的丙型肝炎患者脑损伤的风险增加，这有助于酒精复发的风险。在人类和跨物种中，我们的目标是确定慢性病毒感染和酒精导致免疫细胞功能异常并导致持续性神经精神损害的特定机制。提出了以下具体目标：1)确定丙型肝炎病毒和酒精使用对患有丙型肝炎和急性尿毒症的退伍军人外周T细胞反应和精神功能的影响。生物标本和精神病学数据将来自目前为治疗患有丙型肝炎和AUDS的退伍军人而收集的标本和精神病学数据。我们将获得外周血单个核细胞，并在不同时间对它们进行评估：I)T细胞群的表型变化，II)细胞因子的表面和细胞内聚集，III)对神经抗原和其他抗原的免疫反应性。血液样本将用于测量关键的细胞因子和趋化因子、病毒载量和肝酶。还将使用衡量抑郁、焦虑和饮酒的评级标准。2)调查慢性酒精暴露在调节外周和中枢T细胞反应、中枢神经系统免疫病理以及感染淋巴细胞性脉络膜脑膜炎病毒(LCMV，克隆13变种)的小鼠中的作用，以及焦虑、抑郁和认知障碍的行为迹象。小鼠将长期暴露于乙醇中并依赖于乙醇，然后静脉注射LCMV(或赋形剂)以评估共发病的后果。行为测试将在酒精暴露后进行，以评估焦虑、抑郁样行为和认知功能。将采集血液、脑和脾样本，以：i)测量血液乙醇浓度、肝酶、病毒滴度以及关键细胞因子和趋化因子，ii)评估T细胞频率，包括LCMV特异性CD8 T细胞(利用四聚体分析；H-2LD限制性NP118)，iii)计算产生关键细胞因子(如肿瘤坏死因子)的CD4、CD8和抗原特异性CD8 T细胞的百分比，iv)评估对神经抗原的免疫反应性，以及v)评估神经炎症和神经元变性。研究酒精在调节LCMV感染小鼠的病毒持久性和中枢神经系统免疫病理中的作用将有助于更全面地理解AUDS和丙型肝炎病毒的共病，并可能为未来的治疗开发确定靶点。如果我们的假设得到支持，未来的研究将测试我们的免疫治疗策略，我们已经发现这种策略可以减少神经炎症，并改善甲基苯丙胺依赖小鼠的认知功能。成功治疗酒精引起的神经精神损害的干预措施也很有可能降低复发率并改善治疗结果。

DESCRIPTION (provided by applicant): PROJECT SUMMARY One of the major consequences of chronic alcohol use in brain is increased inflammation that leads to neurodegeneration with associated cognitive and psychiatric impairments. Neuropsychiatric impairments persist in patients following substance abuse and are associated with poorer treatment outcomes. HCV is also associated with a variety of extrahepatic syndromes, including central nervous system (CNS) damage and neuropsychiatric impairments. However, it is unclear whether such cognitive and mood disturbances are a function of systemic disease, damaged hepatic function, or virus infection of the CNS. Animal models are needed to provide new insights into the molecular mechanisms and pathways affected by co-morbid alcohol dependence and chronic viral infection, and also those which are responsible for the persistence of neuropsychiatric impairments following abstinence and viral clearance. Our overall hypothesis is that HCV patients with co-morbid AUDs are at increased risk of brain damage that contributes to alcohol relapse risk. In humans and across species, our goal is to identify specific mechanisms by which chronic viral infection and alcohol induce abnormalities in immune cell function and contribute to persistent neuropsychiatric impairments. The following specific aims are proposed: 1) Determine the effects of HCV and alcohol use on peripheral T cell response and psychiatric function in veterans with co-morbid HCV and AUDs. Biological specimens and psychiatric data will be derived from those currently being collected for a treatment trial in veterans with HCV and co-morbid AUDs. We will obtain peripheral blood mononuclear cells and evaluate them across time for: i) phenotypic changes in T-cell populations, ii) surface and intracellular accumulation of cytokines, and iii) immunoreactivity to neuroantigens and other antigens. Blood samples will be used to measure key cytokines and chemokines, viral load, and liver enzymes. Rating scales that measure depression, anxiety, and alcohol consumption will also be used. Results from the immunoassays will be analyzed in relation to psychiatric measures, viral load, and alcohol use, as well as in relation to the findings from Aim 2. 2) Investigate the role of chronic alcohol exposure in regulating peripheral and central T-cell responses, CNS immunopathology, and behavioral signs of anxiety, depression, and cognitive impairments in mice infected with lymphocytic choriomeningitis virus (LCMV, clone 13 variant), an established model for HCV infections in humans. Mice will be chronically exposed to and dependent on ethanol administered intragastrically followed by intravenous injection of LCMV (or vehicle) to evaluate the consequences of co-morbidity. Behavioral tests will be conducted following ethanol exposure to assess anxiety, depressive-like behavior, and cognitive function. Blood, brain, and spleen samples will be collected to: i) measure blood ethanol concentrations, liver enzymes, viral titers, and key cytokines and chemokines, ii) evaluate T cell frequencies, including LCMV-specific CD8+ T cells (utilizing tetramer analysis; H-2Ld-restricted NP118), iii) calculate the percentage of CD4+, CD8+, and antigen-specific CD8+ T cells producing key cytokines (e.g., TNF- ), iv) evaluate immunoreactivity to neuroantigens, and v) assess neuroinflammation and neuronal degeneration. Examining the role of alcohol in regulating viral persistence and CNS immunopathology in LCMV-infected mice will lead to a more comprehensive understanding of co-morbid AUDs and HCV and may identify targets for future therapeutic development. If our hypotheses are supported, future studies will test our immunotherapeutic strategy that we have found reduces neuroinflammation and improves cognitive function in mouse models of methamphetamine dependence. Interventions that successfully treat alcohol induced neuropsychiatric impairments have a high likelihood of also reducing relapse rates and improving treatment outcomes.